The previously uncharacterized RnpM (YlxR) protein modulates the activity of ribonuclease P in Bacillus subtilis in vitro.

Even though Bacillus subtilis is one of the most studied organisms, no function has been identified for about 20% of its proteins. Among these unknown proteins are several RNA- and ribosome-binding proteins suggesting that they exert functions in cellular information processing. In this work, we have investigated the RNA-binding protein YlxR.

Characterization of RNA-based and protein-only RNases P from bacteria encoding both enzyme types.

A small group of bacteria encode two types of RNase P, the classical ribonucleoprotein (RNP) RNase P as well as the protein-only RNase P HARP (omolog of Nase ). We characterized the dual RNase P activities of five bacteria that belong to three different phyla. All five bacterial species encode functional RNA (gene ) and protein (gene ) subunits of RNP RNase P, but only the HARP of the thermophile (phylum Thermodesulfobacteria) was found to have robust tRNA 5'-end maturation activity in vitro and in vivo in an RNase P depletion strain.

RNA inhibits dMi-2/CHD4 chromatin binding and nucleosome remodeling.

The ATP-dependent nucleosome remodeler Mi-2/CHD4 broadly modulates chromatin landscapes to repress transcription and to maintain genome integrity. Here we use individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) to show that Drosophila Mi-2 associates with thousands of mRNA molecules in vivo. Biochemical data reveal that recombinant dMi-2 preferentially binds to G-rich RNA molecules using two intrinsically disordered regions of unclear function.

Comparative study on tertiary contacts and folding of RNase P RNAs from a psychrophilic, a mesophilic/radiation-resistant, and a thermophilic bacterium.

In most bacterial type A RNase P RNAs (P RNAs), two major loop-helix tertiary contacts (L8-P4 and L18-P8) help to orient the two independently folding S- and C-domains for concerted recognition of precursor tRNA substrates. Here, we analyze the effects of mutations in these tertiary contacts in P RNAs from three different species: (i) the psychrophilic bacterium (), (ii) the mesophilic radiation-resistant bacterium () and (iii) the thermophilic bacterium (). We show by UV melting experiments that simultaneous disruption of these two interdomain contacts has a stabilizing effect on all three P RNAs.

Minimal and RNA-free RNase P in .

RNase P is an essential tRNA-processing enzyme in all domains of life. We identified an unknown type of protein-only RNase P in the hyperthermophilic bacterium : Without an RNA subunit and the smallest of its kind, the 23-kDa polypeptide comprises a metallonuclease domain only. The protein has RNase P activity in vitro and rescued the growth of and strains with inactivations of their more complex and larger endogenous ribonucleoprotein RNase P.

Protein-only RNase P function in Escherichia coli: viability, processing defects and differences between PRORP isoenzymes.

The RNase P family comprises structurally diverse endoribonucleases ranging from complex ribonucleoproteins to single polypeptides. We show that the organellar (AtPRORP1) and the two nuclear (AtPRORP2,3) single-polypeptide RNase P isoenzymes from Arabidopsis thaliana confer viability to Escherichia coli cells with a lethal knockdown of its endogenous RNA-based RNase P. RNA-Seq revealed that AtPRORP1, compared with bacterial RNase P or AtPRORP3, cleaves several precursor tRNAs (pre-tRNAs) aberrantly in E.

Analysis of the Cleavage Mechanism by Protein-Only RNase P Using Precursor tRNA Substrates with Modifications at the Cleavage Site.

Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5'-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein-only RNase P (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here, we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana (AtPRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, with either Rp- or Sp-phosphorothioate, or 2'-deoxy, 2'-fluoro, 2'-amino, or 2'-O-methyl substitutions.

Substrate recognition and cleavage-site selection by a single-subunit protein-only RNase P.

RNase P is the enzyme that removes 5' extensions from tRNA precursors. With its diversity of enzyme forms-either protein- or RNA-based, ranging from single polypeptides to multi-subunit ribonucleoproteins-the RNase P enzyme family represents a unique model system to compare the evolution of enzymatic mechanisms. Here we present a comprehensive study of substrate recognition and cleavage-site selection by the nuclear single-subunit proteinaceous RNase P PRORP3 from Arabidopsis thaliana.

3'-UTRs as a source of regulatory RNAs in bacteria.

Bacterial small non-coding RNAs, sRNAs, have up to now been identified primarily in intergenic regions. Chao et al reveal that the 3'-region of mRNAs is another rich reservoir of sRNAs.

tRNA processing by protein-only versus RNA-based RNase P: kinetic analysis reveals mechanistic differences.

In Arabidopsis thaliana, RNase P function, that is, endonucleolytic tRNA 5'-end maturation, is carried out by three homologous polypeptides ("proteinaceous RNase P" (PRORP) 1, 2 and 3). Here we present the first kinetic analysis of these enzymes. For PRORP1, a specificity constant (k(react)/K(m(sto))) of 3×10(6) M(-1) min(-1) was determined under single-turnover conditions.

Characterization of RNase P RNA activity.

The principle task of the ubiquitous enzyme RNase P is the generation of mature tRNA 5'-ends by removing precursor sequences from tRNA primary transcripts (Trends Genet 19:561-569, 2003; Crit Rev Biochem Mol Biol 41:77-102, 2006; Trends Biochem Sci 31:333-341, 2006). In Bacteria, RNase P is a ribonucleoprotein composed of two essential subunits: a catalytic RNA subunit (P RNA; 350-400 nt) and a single small protein cofactor (P protein; ∼14 kDa). In vitro, bacterial P RNA can catalyze tRNA maturation in the absence of the protein cofactor at elevated concentrations of mono- and divalent cations (Cell 35:849-857, 1983).

Archaeal-bacterial chimeric RNase P RNAs: towards understanding RNA's architecture, function and evolution.

The higher protein content of archaeal RNase P (1 RNA+4 proteins) compared to the bacterial homologue (1 RNA+1 protein) correlates with a large loss of RNA-alone activity (i.e., in the absence of protein cofactors).

A single Arabidopsis organellar protein has RNase P activity.

The ubiquitous endonuclease RNase P is responsible for the 5' maturation of tRNA precursors. Until the discovery of human mitochondrial RNase P, these enzymes had typically been found to be ribonucleoproteins, the catalytic activity of which is associated with the RNA component. Here we show that, in Arabidopsis thaliana mitochondria and plastids, a single protein called 'proteinaceous RNase P' (PRORP1) can perform the endonucleolytic maturation of tRNA precursors that defines RNase P activity.

The making of tRNAs and more - RNase P and tRNase Z.

Transfer-RNA (tRNA) molecules are essential players in protein biosynthesis. They are transcribed as precursors, which have to be extensively processed at both ends to become functional adaptors in protein synthesis. Two endonucleases that directly interact with the tRNA moiety, RNase P and tRNase Z, remove extraneous nucleotides on the molecule's 5'- and 3'-side, respectively.

Analysis of RNase P protein (rnpA) expression in Bacillus subtilis utilizing strains with suppressible rnpA expression.

Bacterial RNase P is composed of an RNA subunit and a single protein subunit (encoded by the rnpB and rnpA genes, respectively). We constructed Bacillus subtilis mutant strains that conditionally express the RNase P protein under control of the xylose promoter (P(xyl)). In one strain (d7), rnpA expression was efficiently repressed in the absence of the inducer xylose, leading to cell growth arrest.

An unusual mechanism of bacterial gene expression revealed for the RNase P protein of Thermus strains.

The RNase P protein gene (rnpA) completely overlaps the rpmH gene (encoding ribosomal protein L34) out of frame in the thermophilic bacterium Thermus thermophilus. This results in the synthesis of an extended RNase P protein (C5) of 163 aa and, by inference, of 240 aa in the related strain Thermus filiformis. Start codons of rnpA and rpmH, apparently governed by the same ribosome binding site, are separated by only 4 nt, which suggests a regulatory linkage between L34 and C5 translation and, accordingly, between ribosome and RNase P biosynthesis.